Delivery of AS-oligonucleotide microspheres to induce dendritic cell tolerance for the treatment of autoimmune type 1 diabetes

ABSTRACT

AS-oligonucleotides are delivered in microsphere form in order to induce dendritic cell tolerance, particularly in the non-obese-diabetic (NOD) mouse model. The microspheres incorporate antisense (AS) oligonucleotides. A process includes using an antisense approach to prevent an autoimmune diabetes condition in NOD mice in vivo and in situ. The oligonucleotides are targeted to bind to primary transcripts CD40, CD80, CD86 and their combinations.

CROSS REFERENCES TO RELATED APPLICATION

Provisional Patent Application Ser. No. 60/570,273, filed May 12, 2004and Provisional Patent Application Ser. No. 60/625,483 filed Nov. 5,2004.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to microsphere delivery ofAS-oligonucleotides in order to induce dendritic cell tolerance,particularly in the non-obese-diabetic (NOD) mouse model. Moreparticularly, the invention relates to drug delivery technology by wayof microspheres that are fabricated using totally aqueous conditions,which microspheres incorporate antisense (AS) oligonucleotides. Thesemicrospheres are used for an antisense approach to prevent an autoimmunediabetes condition in NOD mice in vivo and in situ.

2. Background of the Invention

Microparticles, microspheres, and microcapsules are solid or semi-solidparticles having a diameter of less than one millimeter, more preferablyless than 100 microns, which can be formed of a variety of materials,including synthetic polymers, proteins, and polysaccharides.Microspheres have been used in many different applications, primarilyseparations, diagnostics, and drug delivery.

A number of different techniques can be used to make these microspheresfrom synthetic polymers, natural polymers, proteins and polysaccharides,including phase separation, solvent evaporation, emulsification, andspray drying. Generally the polymers form the supporting structure ofthese microspheres, and the drug of interest is incorporated into thepolymer structure. Exemplary polymers used for the formation ofmicrospheres include homopolymers and copolymers of lactic acid andglycolic acid (PLGA) as described in U.S. Pat. No. 5,213,812 to Ruiz,U.S. Pat. No. 5,417,986 to Reid et al., U.S. Pat. No. 4,530,840 to Ticeet al., U.S. Pat. No. 4,897,268 to Tice et al., U.S. Pat. No. 5,075,109to Tice et al., U.S. Pat. No. 5,102,872 to Singh et al., U.S. Pat. No.5,384,133 to Boyes et al., U.S. Pat. No. 5,360,610 to Tice et al., andEuropean Patent Application Publication Number 248,531 to SouthernResearch Institute; block copolymers such as tetronic 908 and poloxamer407 as described in U.S. Pat. No. 4,904,479 to Illum; andpolyphosphazenes as described in U.S. Pat. No. 5,149,543 to Cohen et al.Microspheres produced using polymers such as these exhibit a poorloading efficiency and are often only able to incorporate a smallpercentage of the drug of interest into the polymer structure.Therefore, substantial quantities of microspheres often must beadministered to achieve a therapeutic effect.

Spherical beads or particles have been commercially available as a toolfor biochemists for many years. For example, antibodies conjugated tobeads create relatively large particles specific for particular ligands.The large antibody-coated particles are routinely used to crosslinkreceptors on the surface of a cell for cellular activation, are bound toa solid phase for immunoaffinity purification, and may be used todeliver a therapeutic agent that is slowly released over time, usingtissue or tumor-specific antibodies conjugated to the particles totarget the agent to the desired site.

One disadvantage of the microparticles or beads currently available isthat they are difficult and expensive to produce. Microparticlesproduced by these known methods have a wide particle size distribution,often lack uniformity, and fail to exhibit long term release kineticswhen the concentration of active ingredients is high. Furthermore, thepolymers used in these known methods are dissolved in organic solventsin order to form the microparticles. They must therefore be produced inspecial facilities designed to handle organic solvents. These organicsolvents could denature proteins or peptides contained in themicroparticles. Residual organic solvents could be toxic whenadministered to humans or animals.

In addition, the available microparticles are rarely of a sizesufficiently small to fit through the aperture of the size of needlecommonly used to administer therapeutics or to be useful foradministration by inhalation. For example, microparticles prepared usingpolylactic glycolic acid (PLGA) are large and have a tendency toaggregate. A size selection step, resulting in product loss, isnecessary to remove particles too large for injection. PLGA particlesthat are of a suitable size for injection must be administered through alarge gauge needle to accommodate the large particle size, often causingdiscomfort for the patient.

Generally, many currently available microparticles are activated torelease their contents in aqueous media and therefore must belyophilized to prevent premature release. In addition, particles such asthose prepared using the PLGA system exhibit release kinetics based onboth erosion and diffusion. In this type of system, an initial burst orrapid release of drug is observed. This burst effect can result inunwanted side effects in patients to whom the particles have beenadministered.

Microparticles prepared using lipids to encapsulate target drugs areknown. For example, lipids arranged in bilayer membranes surroundingmultiple aqueous compartments to form particles may be used toencapsulate water soluble drugs for subsequent delivery as described inU.S. Pat. No. 5,422,120 to Sinil Kim. These particles are generallygreater than 10 microns in size and are designed for intra articular,intrathecal, subcutaneous and epidural administration. Alternatively,liposomes have been used for intravenous delivery of small molecules.Liposomes are spherical particles composed of a single or multiplephospholipid and cholesterol bilayers. Liposomes are 30 microns orgreater in size and may carry a variety of water-soluble orlipid-soluble drugs. Liposome technology has been hindered by problemsincluding purity of lipid components, possible toxicity, vesicleheterogeneity and stability, excessive uptake and manufacturing orshelf-life difficulties.

An objective for the medical community is the delivery of nucleic acidsto the cells in an animal for diabetes treatment. For example, nucleicacids can be delivered to cells in culture (in vitro) relativelyefficiently, but nucleases result in a high rate of nucleic aciddegradation when nucleic acid is delivered to animals (in vivo).

In addition to protecting nucleic acid from nuclease digestion, anucleic acid delivery vehicle must exhibit low toxicity, must beefficiently taken up by cells and have a well-defined, readilymanufactured formulation. As shown in clinical trials, viral vectors fordelivery can result in a severely adverse, even fatal, immune responsein vivo. In addition, this method has the potential to have mutageniceffects in vivo. Delivery by enclosing nucleic acid in lipid complexesof different formulations (such as liposomes or cationic lipidcomplexes) has been generally ineffective in vivo and can have toxiceffects. Complexes of nucleic acids with various polymers or withpeptides have shown inconsistent results and the toxicity of theseformulations has not yet been resolved. Nucleic acids also have beenencapsulated in polymer matrices for delivery, but in these cases theparticles have a wide size range and the effectiveness for therapeuticapplications has not yet been demonstrated.

Therefore, there is a need for addressing nucleic acids delivery issues,and there is an on-going need for development of microspheres and to newmethods for making microspheres. Details regarding microspheres arefound in U.S. Pat. No. 6,458,387 to Scott et al., U.S. Pat. Nos.6,268,053, 6,090,925, 5,981,719 and 5,599,719 to Woiszwillo et al., andU.S. Pat. No. 5,578,709 to Woiszwillo. These and all referencesidentified herein are incorporated by reference hereinto.

SUMMARY OF THE INVENTION

In accordance with the present invention, DNA to be delivered todendritic cells is delivered as microspheres. It is believed that such adelivery approach prevents access of the nucleases to the nucleic acidswithin the microsphere. Microsphere delivery of AS-oligonucleotides iscarried out in order to induce dendritic cell tolerance, particularly inthe NOD mouse model. The microspheres are fabricated using aqueousconditions, which microspheres incorporate antisense (AS)oligonucleotides. These microspheres are used to inhibit gene expressionand to prevent an autoimmune diabetes condition in NOD mice in vivo andin situ.

In a preferred aspect of the invention, three AS-oligonucleotidestargeted to the CD40, CD80 and CD86 primary transcripts are synthesized,and an aqueous solution of the oligonucleotide mixture is prepared andcombined with a polymer solution. After processing, microspherescontaining the oligonucleotides are provided, and these are delivered tothe NOD mice.

These and other aspects, objects, features and advantages of the presentinvention, including the various combinations, will be apparent from andclearly understood through a consideration of the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

In the course of this description, reference will be made to theattached drawings, wherein:

FIG. 1 is a schematic illustration of the role of dendritic cells in theautoimmune destruction of pancreatic insulin-producing beta-cells inType 1 diabetes;

FIG. 2 is a diagram of the Beta-Galactosidase gene-containing plasmidvector;

FIG. 3 shows photomicrographs providing evidence for transfection of NIH3T3 fibroblast cells with the plasmid DNA microspheres;

FIG. 4 is a photomicrograph of agarose electrophoresis gel of nakedplasmid DNA and of two plasmid DNA microsphere formulations according tothe invention, each after exposure to DNAase;

FIG. 5 is a bar graph of Beta-Galactosidase activity in four differentplasmid DNA applications.

FIG. 6 is a scanning electron migrograph of microspheres ofAS-oligonucleotides and poly-L-lysine polycation;

FIG. 7 is a scanning electron micrograph of microspheres ofAS-oligonucleotides and poly-L-ornithine polycation; and

FIG. 8 is a plot summarizing diabetes incidence in three groups of NODmice treated with the microspheres and according to other procedures fordelivery of the three primary transcripts.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As required, detailed embodiments of the present invention are disclosedherein; however, it is to be understood that the disclosed embodimentsare merely exemplary of the invention, which may be embodied in variousforms. Therefore, specific details disclosed herein are not to beinterpreted as limiting, but merely as a basis for the claims and as arepresentative basis for teaching one skilled in the art to variouslyemploy the present invention in virtually any appropriate manner.

The preferred embodiment prevents autoimmune insulin-dependent diabetesby formulating and injecting antisense (AS)-oligonucleotide microspheresdescribed herein targeting the primary transcripts of CD40, CD80 andCD86. These oligonucleotides are designed to induce immune tolerance inan attempt to prevent destruction of the insulin producing beta cells inthe NOD mouse model. The events leading to the destruction of these betacells is illustrated in FIG. 1. This illustrates how Type 1 diabetes ismanifested by the autoimmune destruction of the pancreaticinsulin-producing beta cells in the NOD mouse, as well as in humans. Atthe time of clinical onset, humans have 10-20% residual beta cell mass.Sparing of this residual mass can result in remaining insulin levelswhich are adequate to regulate glucose levels. The microparticles of theinvention are provided to interfere with the autoimmune destruction ofthe beta cells which is illustrated in FIG. 1.

It will be appreciated that dendritic cells (DC) can be activated to bepotent antigen presenting cells found in all tissues and which arehighly concentrated under the skin. These antigen presenting dendriticcells function as triggers of the immune response through the activationof T-cells, particularly in lymph nodes.

FIG. 2 is a drawing of a plasmid vector containing theBeta-galactosidase gene that can be used to transfect NIH 3T3 fibroblastcells. In vitro evidence for the transfection of NIH 3T3 fibroblastcells with the plasmid DNA microspheres is shown in FIG. 3 by the cellswhich stain blue in color in response to the addition of theBeta-Galactosidase x-gal(5-bromo-4-chloro-3-indolyl-beta-galactopyranoside) substrate.

FIG. 4 illustrates the ability of microspheres to protect DNA insolution. This is an agarose electrophoresis gel showing nucleaseprotection imparted by microspheres of plasmid DNA produced generally asnoted herein. In the Plasmid samples 1, 2 and 3, naked plasmid DNA wasexposed to DNAse, with the smears indicating plasmid DNA degradation ateach of the three levels of DNAase application. In the Particle 1 andParticle 2 samples, plasmid DNA microsphere formulations were exposed toDNAase. The lack of smearing indicates the microsphere formulations showshielding of the plasmid DNA from degradation. Particle 1 plasmid DNAsamples show enhanced protection over Particle 2 plasmid DNA samples.

FIG. 5 reports on Beta-Galactosidase activity of four different plasmidDNA applications when transfected into cells. The naked plasmid DNAapplication showed very low levels. Somewhat greater levels areindicated for plasmid DNA cationic lipid complex application usinglipofectamine, a commercial cationic lipid, as the delivery vehicle.Substantially greater activity is shown for two pDNA microspheres, withMicrospheres 1 corresponding to Particle 1 of FIG. 4, and Microspheres 2corresponding to Particle 2 of FIG. 4.

In making the microspheres that are used for autoimmune treatment ofdiabetes in mice, three AS-oligonucleotides are dissolved in aqueoussolution and combined with water soluble polymer(s) and a polycation.The solution typically is incubated at about 60-70° C., cooled to about23° C., and the excess polymer is removed. Microspheres are formed whichare believed to contain the three AS-oligonucleotides having thefollowing sequences, wherein an asterisk indicates thioation:

CD 40-AS: 5′C*AC* AG*C C*GA* GG*C* AA*A GA Seq ID 1 *C* AC*C A*T*G C*AG*GG*C* A-3′: CD80-AS: 5′-G*GG* AA*A G*CC* AG*G A*AT* Seq ID 2 CT*A G*AG*CC*A A*TG G*A-3′: CD86-AS: 5′-T*GG* GT*G C*TT* CC*G T*AA* Seq ID 3 GT*TC*TG* GA*A C*AC* G*T*C-3′:

More particularly, the nucleic acids typically comprise between about 30and about 100 weight percent of the microspheres and have an averageparticle size of not greater than about 50 microns. Typically, they areprepared as follows. An aqueous solution of the oligonucleotide mixtureis prepared by combining aliquots from three oligonucleotide solutions,each solution containing one of these three types.

A solution containing the three types of oligonucleotides is prepared.The solutions preferably contain about 10 mg/ml oligonucleotide. Theseare combined with aliquots of a 10 mg/ml stock solution of polycationsolution at volumetric ratios of polycation:oligonucleotide of fromabout 1:1 to about 4:1. Polymer solutions of polyvinyl pyrrolidoneand/or of polyethylene glycol are prepared and combined with the othersolutions. Heating, cooling, centrifuging and washing multiple timesprovide an aqueous suspension which typically is frozen and lyophilizedto form a dry powder of microspheres comprising oligonucleotide andpolycation.

Microspheres according to the invention are a viable non-viral deliverytool for plasmid DNA and antisense oligonucleotides and other nucleicacids. They allow for in vitro delivery of Beta-Galactosidase plasmidDNA in 3T3 fibroblast cells. The microspheres protect plasmid DNA fromnuclease activity. High levels of Beta-Galactosidase activity areexpressed following transfection with the microsphere formulations.

Microspheres containing the antisense oligonucleotides of interestdown-regulate surface cell antigens CD40, CD80 and CD86, known to becritical in the activation of the autoimmune reaction that results indestruction of insulin-producing beta cells of the pancreas. This can beaccomplished by subcutaneous injection to dendritic cells located underthe skin. NOD mice studies demonstrate effective prevention of theautoimmune destruction of beta cells. The DNA and oligonucleotidemicrospheres are effective transfection vehicles in vitro and in vivo.Dendritic cells appear to take up the oligonucleotide microspheres andsuppress the expression of surface cell antigens CD40, CD80 and CD86.The anitsense oligonucleotide microspheres effectively prevent diabetesdevelopment in the NOD mouse.

The following Examples illustrate certain features and advantages of theinvention in order to further illustrate the invention. The Examples arenot to be considered limiting or otherwise restrictive of the invention.

EXAMPLE 1

Three AS-oligonucleotides targeted to the CD40, CD80 and CD86 primarytranscripts were synthesized by the DNA synthesis facility at Universityof Pittsburgh (Pittsburgh, Pa.). The AS-oligonucleotides sequences are:

CD 40-AS: 5′C*AC* AG*C C*GA* GG*C* AA*A GA Seq ID 1 *C* AC*C A*T*G C*AG*GG*C* A-3′: CD80-AS: 5′-G*GG* AA*A G*CC* AG*G A*AT* Seq ID 2 CT*A G*AG*CC*A A*TG G*A-3′: CD86-AS: 5′-T*GG* GT*G C*TT* CC*G T*AA* Seq ID 3 GT*TC*TG* GA*A C*AC* G*T*C-3′:

An aqueous solution of the oligonucleotide mixture was prepared bycombining aliquots of three oligonucleotide solutions, each of whichcontained one type of oligonucleotide, to form a 10 [mg/ml] solution ofthe three types of oligonucleotides. 10 [mg/ml] poly-L-lysine.HBr indiH2O (poly-L-lysine.HBr up to 50,000 by Bachem, King of Prussia, Pa.)was prepared. Poly-L-lysine.HBr was added to the oligonucleotidessolution at a volumetric ratio of 1:1. The mixture was vortexed gently.A 25% polymer solution containing 12.5% PVP (polyvinyl pyrrolidone,40,000 Daltons, Spectrum Chemicals, Gardena, Calif.) and 12.5% PEG(polyethylene glycol, 3,350 Daltons, Spectrum Chemicals, Gardena,Calif.) in 1M Sodium Acetate (Spectrum, Gardena, Calif.) at pH=5.5 wasmade. The polymer solution was added in a 2:1 volumetric ratio asfollows: 750 μl of AS-oligonucleotides, 0.75 ml of poly-L-lysine.HBr,3.0 ml of PEG/PVP, and a total volume of 4.50 ml.

The batch was incubated for 30 minutes at 70° C. and then cooled to 23°C. Upon cooling, the solution became turbid and precipitation occurred.The suspension was then centrifuged, and the excess PEG/PVP was removed.The resulting pellet was washed by resuspending the pellet in deionizedwater, followed by centrifugation and removal of the supernatant. Thewashing process was repeated three times. The aqueous suspension wasfrozen and lyophilized to form a dry powder of microspheres comprisingoligonucleotide and poly-L-lysine.

FIG. 6 presents a scanning electron micrograph (SEM) of the 1:1poly-L-lysine:oligonucleotide ratio material. Microspheres, 0.5-4 μm insize, with an average particle size of approximately 2.5 μm werefabricated. Precipitation of an unknown material was also observed.Additional studies by HPLC determined that the precipitation wascomprised of residual PEG/PVP, mostly PVP.

EXAMPLE 2

AS-oligonucleotides targeted to the CD40, CD80 and CD86 primarytranscripts were the AS-oligonucleotides sequences of Example 1. Anaqueous solution of the oligonucleotide mixture was prepared bycombining aliquots of the three oligonucleotide solutions, each of whichcontained one type of oligonucleotide, to form a 10 [mg/ml] solution ofthe three types of oligonucleotides. A solution of oligonucleotidemixture was prepared. 5 [mg/ml] poly-L-ornithine.HBr in diH₂O(poly-L-ornithine.HBr 11,900 (vis) by Sigma) was prepared.Poly-L-ornithine.HBr was added to the oligonucleotides solution. Themixtures were vortexed gently. A 25% polymer solution containing 12.5%PVP (40,000 Daltons, Spectrum Chemicals, Gardena, Calif.) and 12.5% PEG(3,350 Daltons, Spectrum Chemicals, Gardena, Calif.) in 0.1.M SodiumAcetate (Spectrum Chemicals, Gardena, Calif.) at pH=5.5 was made. Thepolymer solutions were added. Incubation and rinses followed asdescribed in Example 1. 1.5 ml of the AS-oligonucleotides, 1.5 ml of thepoly-L-ornithine.HBr, 3 ml of the PEG/PVP, and a total volume of 6.0 mlwas prepared.

FIG. 7 presents an SEM of this 1:1 poly-L-ornithine:oligonucleotideratio material. Microspheres, 0.2-8 μm in size, with an average particlesize of approximately 2 μm were fabricated. Precipitation of an unknownmaterial was also observed. Additional HPLC studies were able to provethat this precipitation was comprised of residual PEG/PVP, mostly PVP.

EXAMPLE 3

In vivo studies were conducted using the NOD mouse model of Type 1diabetes mellitus. Type 1 diabetes is manifested by the autoimmunedestruction of the pancreatic insulin-producing beta cells asillustrated in FIG. 1. AS-oligonucleotides were used in threeapplications in an attempt to interfere with the autoimmune destructionof beta cells. The goal was to interfere with the dendritic cellfunction by targeting the primary transcripts of CD40, CD80 and CD86,which encode dendritric cell surface proteins required for T-cellactivation. Dendritic cells with low levels of CD40, CD80 and CD86 areknown to promote suppressive immune cell networks in vivo. Thesecascades can result in T-cell hyporesponsiveness to beta cells in vivo.

In the first group of test animals, dendritic cells were propagated exvivo from bone marrow progenitors of NOD mice. Combinations of the threeAS-oligonucleotides targeting the primary transcripts of CD40, CD80 andCD86 were added to the cells in tissue culture. After incubation, theAS-oligonucleotide transfected dendritic cells were injected intosyngenetic recipients of 5 to 8 weeks of age (not yet diabetic). This isa known ex-vivo delivery approach.

In parallel, AS-oligonucleotide microspheres were injected directly intoother NOD mice of the same age. A single injection was carried out oneach thus-treated mouse. Another group of these NOD mice was not treatedand served as a control.

FIG. 8 shows that the control, untreated NOD mice all developed diabetesby age 23 weeks. The ex vivo AS-oligonucleotide transfected andre-infused dendritic cells group (AS-ODN DC) showed delayed developmentof diabetes, with 20% remaining “Diabetes Free”, indicating glucoselevels are maintained within a non-diabetic range. Of the microspheresin vivo-injected NOD mice, 71% remained “Diabetes Free” at 43 weeks.

It will be understood that the embodiments of the present inventionwhich have been described are illustrative of some of the applicationsof the principles of the present invention. Numerous modifications maybe made by those skilled in the art without departing from the truespirit and scope of the invention. Various features which are describedherein can be used in any combination and are not limited to precisecombinations which are specifically outlined herein.

1. A composition that comprises microspheres comprising oligonucleotidesfor treatment of type 1 diabetes, wherein said microspheres contain afirst antisense sequence that targets a primary transcript of CD40, asecond antisense sequence that targets a primary transcript of CD80, anda third antisense sequence that targets a primary transcript of CD86,wherein each of said first, second and third oligonucleotides reduces orsuppresses in vivo expression of CD40, CD80 and CD86 respectively, andwherein said oligonucleotides comprise greater than about 30 weightpercent of the microspheres, based on the total weight of themicrospheres, said microspheres having an average particle size of notgreater than about 50 microns and at least 0.2 to 8 microns, and saidmicrospheres, when administered, treat type 1 diabetes.
 2. A process fordelivering nucleic acids in the form of microspheres to an individualwith type I diabetes, comprising administering to said individualmicrospheres in accordance with claim 1, by intravenous, intramuscular,subcutaneous, topical, intradermal, intraperitoneal, oral, pulmonary,ocular, nasal or rectal routes.
 3. A process for protecting beta cellsof the pancreas of non-obese diabetic mice from autoimmune destruction,comprising subcutaneously injecting microspheres in accordance withclaim
 1. 4. A process for protecting beta cells of the pancreas ofindividuals from autoimmune destruction, comprising subcutaneouslyinjecting microspheres in accordance with claim
 1. 5. A process forprotecting beta cells of the pancreas of humans from autoimmunedestruction, comprising subcutaneously injecting microspheres inaccordance with claim
 1. 6. A process for protecting beta cells of thepancreas of individuals from autoimmune destruction and onset of type 1diabetes comprising subcutaneously injecting microspheres in accordancewith claim
 1. 7. A pharmaceutical composition that comprisesmicrospheres wherein said microspheres contain a first antisenseoligonucleotide sequence that targets a primary transcript of CD40, asecond antisense oligonucleotide sequence that targets a primarytranscript of CD80, and a third antisense oligonucleotide sequence thattargets a primary transcript of CD86, wherein each of said first, secondand third antisense oligonucleotides reduces or suppresses theexpression of CD40, CD80 and CD86 respectively, and wherein theantisense oligonucleotides comprise greater than about 30 weight percentof the microspheres.
 8. The composition of claim 7 wherein the antisenseoligonucleotides comprise greater than 60% by weight of themicrospheres.
 9. The composition of claim 7, wherein the microspheresfurther comprise a polycation.
 10. The composition of claim 9, whereinthe microspheres consist essentially of antisense oligonucleotides andthe polycation.
 11. The composition of claim 7, wherein the microspheresare capable of being uptaken by dendritic cells.
 12. The composition ofclaim 7, wherein the composition is an injectable composition suitablefor in vivo delivery.
 13. The composition of claim 12, wherein thecomposition is more effective than a composition that comprises ex-vivodendritic cells transfected with antisense oligonucleotides comprisingSEQ ID NOS: 1-3 in treatment of type 1 diabetes.
 14. The composition ofclaim 12, wherein the composition is suitable for subcutaneousadministration.
 15. The composition of claim 7, wherein the antisenseoligonucleotides comprise SEQ ID NOS: 1-3.
 16. The composition of claim7 wherein the microspheres have an average particle size less than about50 microns and at least 0.2 to 8 microns.
 17. An injectable compositionfor the treatment of type 1 diabetes comprising microspheres, saidmicrospheres comprising a first antisense oligonucleotide sequence thattargets a primary transcript of CD40, a second antisense oligonucleotidesequence that targets a primary transcript of CD80, and a thirdantisense oligonucleotide sequence that targets a primary transcript ofCD86, wherein each of said first, second and third antisenseoligonucleotides reduces or suppresses the expression of CD40, CD80 andCD86 respectively, wherein said first, second and third antisenseoligonucleotides comprise greater than about 30% by weight of saidmicrospheres, and said microspheres, when administered, treat type 1diabetes.
 18. The composition of claim 17, wherein the compositioncomprises a mixture of three different antisense oligonucleotides. 19.The composition of claim 17, wherein the antisense oligonucleotidescomprise SEQ ID NOS: 1-3.
 20. The composition of any of claim 1, 7 or 17wherein the microspheres have a particle size of 0.2 microns to 8microns.
 21. The composition of any of claim 1, 7 or 17 wherein themicrospheres have a particle size of 0.5 microns to 4 microns.
 22. Thecomposition of any of claim 1, 7 or 17 wherein the microspheres have anaverage particle size of about 2 microns.
 23. The composition of any ofclaim 1, 7 or 17 wherein the microspheres have an average particle sizeof about 2.5 microns.